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United States Department of Agriculture

Agricultural Research Service

Research Project: EPIDEMIOLOGY, ECOLOGY, AND MOLECULAR GENETICS OF ANTIMICROBIAL RESISTANCE IN PATHOGENIC AND COMMENSAL BACTERIA FROM FOOD ANIMALS

Location: Bacterial Epidemiology and Antimicrobial Resistance

Title: Development of a High-Throughput Multiplex PCR and Capillary Electrophoresis Technique for Serotype Determination of Salmonella Enterica Food Animal Isolates

Authors
item Frye, Jonathan
item Leader, Brandon - WA DOH, SHORELINE, WA
item Cray, Paula
item Hu, Jinxin - WA DOH, SHORELINE, WA
item Boyle, David - WA DOH, SHORELINE, WA

Submitted to: Proceedings of the International Conference on Emerging Infectious Diseases
Publication Type: Abstract Only
Publication Acceptance Date: February 4, 2008
Publication Date: March 16, 2008
Citation: Frye, J.G., Leader, B.T., Cray, P.J., Hu, J., Boyle, D.S. 2008. Development of a High-Throughput Multiplex PCR and Capillary Electrophoresis Technique for Serotype Determination of Salmonella Enterica Food Animal Isolates. Proceedings of the International Conference on Emerging Infectious Diseases. March 16-19, 2008. Atlanta, GA. 184.

Technical Abstract: Background: Previously, a multiplex PCR technique was developed to identify the top 30 human clinical serotypes of Salmonella enterica. To improve the speed, ease of use, utility and discriminatory ability of the technique, additional primers were added and the PCR product discrimination and analysis was automated by capillary electrophoresis. Methods: Fifteen genes, whose distribution reflects the different serotypes of Salmonella enterica, were targeted for amplification in a single multiplex PCR reaction. All forward primers incorporated a universal sequence complementary to a carboxyfluorescein (FAM) linked universal primer used to label all products for detection. The primer pairs and the universal primer were combined in a master mix containing a Hot Start Taq polymerase (Bioline, USA Inc, Randolph, MA, USA). Templates were prepared by the boiled colony method and thermocycling parameters were 94°C 15 min, (94°C 30s, 57°C 90s, 72°C 30s) x 25, 72°C 5min, (94°C 30s, 68°C 90s, 72°C 30s) x 15, 72°C 5min. Samples were diluted 1:100 (v/v) in formamide containing carboxy-X-rhodamine (ROX) labeled GENEFLO 625 DNA Ladder (CHIMERx, Milwaukee, WI, USA) and separated in an ABI 3100 Avant gene analyzer on a 50 cm capillary. Results: Fifty-four Salmonella isolated from raw pork were analyzed by the multiplex PCR analysis technique (PCR) and by traditional serotyping (TS). PCR resulted in serotypes for 49/54 isolates, while TS resulted in 53/54 serotypes with one failing identification by both techniques. Results were the same for the 49 isolates that were serotyped by both techniques, (28 serotype Typhimurium, 18 Infantis, and 3 Seftenberg). Traditional serotype determination also identified four isolates as serotype Muenchen. Overall use of PCR resulted in a 92% (49/53) accuracy, lower cost (~$5 for PCR versus ~$39 for TS), and quicker turn-around time (6 h for PCR versus weeks for TS) as compared to TS. Conclusions: Automated multiplex PCR successfully determined the serotype of Salmonella enterica isolated from pork 92% of the time as compared to traditional serotyping indicating that a rapid and more economical alternative to TS is available. The inclusion of additional primers will increase the identification of additional serotypes.

Last Modified: 8/1/2014